We find that the interaction

We find that the interaction of NSF and SNAP is critically involved in NMDAR-induced PICK1 unclustering. The binding of SNAP to NSF increases the ATPase activity of NSF, which is known to be essential for the unbinding of PICK1 from GluR2 (Hanley et al., 2002). Disruption of the association between NSF and SNAP with an interfering peptide significantly inhibited the NMDA/low Mg2+-induced redistribution of PICK1 suggesting the importance of this interaction. However, the incomplete blockade we observe indicates that the basal ATPase activity of NSF that is independent of SNAP is likely to be sufficient to mediate some PICK1 unclustering. In the central nervous system, NO is synthesized by the nitric oxide synthase (NOS), a calcium/calmodulin-dependent enzyme. NOS complexes with NMDARs via an interaction with PSD-95 (Christopherson et al., 1999), which likely facilitates calcium influx-mediated NO synthesis (Bredt and Snyder, 1989). We have previously shown that NMDA/low Mg2+ treatment allows for a high level calcium entry, and that the unclustering of PICK1 is dependent on intracellular calcium concentrations. If this calcium is acting directly through the activation of NOS, inhibition of calmodulin activity would be expected to block NMDAR-mediated PICK1 unclustering. However, we found that calmodulin inhibitors themselves affect the basal clustering of PICK1 in diacerein (data not shown). This suggests that calmodulin-dependent effectors may influence PICK1 clustering at multiple levels, although we can not confirm its role in activating NOS in our experiments. The role of NO in NMDAR-dependent long-term plasticity in the CA1 region of the hippocampus remains in dispute. At one time evidence suggested that long-term plasticity at CA1 hippocampal synapses was expressed via the retrograde action of postsynaptically generated nitric oxide (NO) on presynaptic terminals (O'Dell et al., 1991, Schuman and Madison, 1991), where it increases cGMP levels and influences vesicular release (Stevens, 1988). Additional evidence suggests that NO can act presynaptically to drive synaptic depression (Gage et al., 1997). However, subsequent debate challenged whether NO is actually involved in the long-term potentiation (LTP) of synaptic responses as it was found that LTP was obtainable in the absence of NOS (O'Dell et al., 1994). Another study furthered the controversy by showing that LTP is blocked in independently created NOS mutant mice (Kantor et al., 1996). While the role for NO in potentiation remains unresolved, intriguing recent evidence shows a role for postsynaptic NO following chemical LTP, where S-nitrosylation of NSF increases AMPAR trafficking to the membrane surface (Huang et al., 2005). This together with our findings suggests that NO may contribute to the potentiation of synaptic responses. Our results lie in apparent conflict with studies suggesting no role for NO in LTP. This conflict may be explained by the existence of multiple mechanisms contributing to synaptic potentiation. Standard models of NMDAR-LTP suggest that insertion of GluR1-containing receptors, which do not bind NSF, mediate LTP in a CaMKII-dependent manner (Hayashi et al., 2000, Passafaro et al., 2001, Plant et al., 2006). These receptors are then thought to be replaced by cycling GluR2/3 AMPARs (Plant et al., 2006). Our data showing a preferential increase in surface GluR2 over GluR1 receptors could reflect the later effects of this process of receptor exchange. Alternatively, as classically defined NMDAR-LTP likely occurs independently of NO-mediated effects, the potentiation described here may reflect a different form of plasticity. The release of NO by NMDAR activation may mediate the delivery of previously internalized GluR2/3-containing (GluR1-lacking) AMPARs held intracellularly by PICK1, a process more consistent with a model of dedepression. As NMDAR-dependent LTP and dedepression can be elicited by the same stimuli, the preexisting state of synapses (Montgomery and Madison, 2004) may determine whether or not synaptic potentiation elicited is NO-dependent.